M2-associated Genes Research Articles

Serum Amyloid A1 Induces Classically Activated Macrophages: A Role for Enhanced Fibril Formation.

AA amyloidosis belongs to the group of amyloid diseases which can follow chronic inflammatory conditions of various origin. The disease is characterized by the deposition of insoluble amyloid fibrils formed by serum amyloid A1 (SAA1) leading eventually to organ failure. Macrophages are intimately involved in the fibrillogenesis as well as in the clearance of amyloid fibrils. In vivo, macrophages may occur as classically (M1) or alternatively activated (M2) macrophages. We investigate here how SAA1 might affect the macrophage phenotype and function. Gene microarray analysis revealed upregulation of 64 M1-associated genes by SAA1. M1-like polarization was further confirmed by the expression of the M1-marker MARCO, activation of the NF-κB transcription factor, and secretion of the M1-cytokines TNF-α, IL-6, and MCP-1. Additionally, we demonstrate here that M1-polarized macrophages exhibit enhanced fibrillogenic activity towards SAA1. Based on our data, we propose reconsideration of the currently used cellular amyloidosis models towards an in vitro model employing M1-polarized macrophages. Furthermore, the data suggest macrophage repolarization as potential intervention strategy in AA amyloidosis.

Effects of Macrophage Colony-Stimulating Factor Isoforms on Leukemia Progression in Mouse Acute Myeloid Leukemia Model

Oncogenic events occur by a variety of mechanisms, including point mutations, in-frame deletions or insertions, epigenetic modifications or even surrounding changes. As an important growth factor, M-CSF plays different roles in complicated pathological context and there are complex heterogeneities between M-CSF different splicing isoforms (sM-CSF and mM-CSF). The role of M-CSF and the difference between the two isoforms in hematological malignancies, which are significantly different from solid tumors, is poorly understood. In this study, we studied the different effects of mM-CSF and sM-CSF on both intrinsic factor-leukemia cells and surrounding linker-macrophage in acute myeloid leukemia mice model.The high level expression of M-CSF was found in myeloid leukemia patients from bloodspot database. We established MLL-AF9 induced AML mouse model with high level expression of sM-CSF or mM-CSF. We found mM-CSF could delay the progression of leukemia. Subsequently, we investigated the effect of the two isoforms on both leukemia cells and macrophages. More proliferative cells were detected in leukemia cells with high level M-CSF isoforms. In vitro colony assay experiments showed sM-CSF could promote the self-renew of leukemia cells. Morphological and flow cytometry analysis indicated M-CSF isoforms had different potential in promoting leukemia cell differentiation. Moreover, myeloid differentiation associated transcription factor-Cebpe and Runx1 might account for differentiation promoting effect by mM-CSF. As for macrophages, M-CSF isoforms could increase monocytes and macrophages in leukemic microenvironment by both growth-promoting and recruitment effects. Additionally, mM-CSF promoted the expression of both M1 and M2-associated genes. Meanwhile mM-CSF promoted the specific phagocytosis potential of macrophages to leukemia cells. Furthermore, the gene expression profiles of LAMs (leukemia associated macrophages) revealed mM-CSF did make macrophages a special phenotype with enhanced oxidative phosphorylation, metabolism and phagocytosis pathway. Moreover, phagocytosis of LAMs educated by mM-CSF may be mediated by oxidative phosphorylation.Our results suggested that the diverse effects of M-CSF two isoforms in leukemic microenvironment and mM-CSF delayed the leukemia course by affecting both leukemia cell differentiation and macrophage frequencies along with specific phagocytosis to leukemia cells. The work reveals that both the intrinsic and surrounding factors participate in tumor progression and the complex of carcinogenesis and the interaction between tumor cell itself and microenvironment linkers should be further studied.This work was supported by Grants 81370634 and 81570153 from the National Natural Science Foundation of China (NSFC); program 2016-I2M-2-006 from CAMS Initiative for Innovative Medicine; grant 17JCZDJC35000 from the Tianjin Science and Technology Committee. DisclosuresNo relevant conflicts of interest to declare.

17β-Estradiol Reduces Demyelination in Cuprizone-fed Mice by Promoting M2 Microglia Polarity and Regulating NLRP3 Inflammasome

Estrogen produces a beneficial role in animal models of multiple sclerosis (MS). The effect of 17β-estradiol therapy on microglia polarization and neuroinflammation in the corpus callosum of the cuprizone-induced demyelination model has not been elucidated. In this study, mice were given 0.2% cuprizone (CPZ) for 5 weeks to induce demyelination during which they received 50 ng of 17β-estradiol (EST), injected subcutaneously in the neck region, twice weekly. Data revealed that treatment with 17β-estradiol therapy (CPZ+EST) improved neurological behavioral deficits, displayed by a significant reduction in escape latencies, in comparison to untreated CPZ mice. Also, administration of 17β-estradiol caused a decrease in demyelination levels and axonal injury, as demonstrated by staining with Luxol fast blue, immunofluorescence to myelin basic protein, and transmission electron microscopy analysis. In addition, at the transcriptional level in the brain, mice treated with 17β-estradiol (CPZ+EST) showed a decrease in the levels of M1-assosicted microglia markers (CD86, iNOS and MHC-II) whereas M2-associated genes (Arg-1, CD206 and Trem-2) were increased, compared to CPZ mice. Moreover, administration of 17β-estradiol resulted in a significant reduction (∼3-fold) in transcript levels of NLRP3 inflammasome and its downstream product IL-18, compared to controls. In summary, this study demonstrated for the first time that exogenous 17β-estradiol therapy robustly leads to the reduction of M1 phenotype, stimulation of polarized M2 microglia, and repression of NLRP3 inflammasome in the corpus callosum of CPZ demyelination model of MS. The positive effects of 17β-estradiol on microglia and inflammasome seems to facilitate and accelerate the remyelination process.

Abstract 2836: Expression of the DEK oncogene in breast cancer cells promotes M2 polarization of tumor associated macrophages

Abstract Breast cancer is the second leading cause of cancer deaths among women. DEK is a known oncoprotein found to be highly expressed in more than 60% breast cancers and is found to be an independent marker of poor prognosis. However, the molecular mechanisms by which DEK promotes tumor progression are poorly understood. To elucidate the oncogenic functions of DEK, we performed RNA-seq analysis on isogenic Dek-knockout and complemented murine breast cancer cells, which indicated dysregulation of immune signaling. Among the target genes identified and confirmed was an upregulation of thymic stromal lymphopoietin (TSLP) in Dek expressing murine breast cancer cells. TSLP was previously shown by Han et al to amplify the alternative (M2) activation of macrophages. M2-like macrophages are tumor promoting, they recycle iron for cell growth and encourage tissue remodeling and repair activities like angiogenesis. To test the immune modulating functions of Dek-expressing breast cancer cells, we treated bone marrow derived macrophages (BMDM) with tumor cell conditioned media. We found that in vitro, Dek expressing cancer cells induced an M2-like polarization of macrophages, as determined by the expression of M2-associated genes, enhanced migration, and iron recycling phenotypes, which was accompanied by inhibited ERK1/2 signaling. We found this phenotypic trend to be true in vivo, as well. In sectioned mammary tumors from MMTV-RontgDek+/+ and MMTV-RontgDek−/− mice, we see lower levels of iron staining in Dek+/+ tumors than in Dek−/− tumors using Prussian blue staining that co-localized with F4/80 macrophage marker staining. This suggested that Dek expression in the tumors induced an iron recycling, M2-like phenotype in tumor associated macrophages. Furthermore, we observed increased TSLP expression and angiogenesis, as determined by CD31 staining, in Dek-expressing tumors in vivo. These findings suggest that tumor Dek expression may promote breast cancer progression by inducing M2-like macrophage polarization in a murine breast cancer model. Citation Format: Miranda Shephard, Nicholas Pease, Lisa M. Privette Vinnedge. Expression of the DEK oncogene in breast cancer cells promotes M2 polarization of tumor associated macrophages [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2836.

Abstract 4556: Targeting the TAM receptors on prostate cancer tumor-associated macrophages

Abstract Tumor growth and progression is influenced by the composition of the tumor microenvironment including host stromal cells and immune cells. In prostate cancer, a large proportion of the tumor volume is made up of macrophages, and high macrophage infiltrate correlates with poor prognosis. Macrophages can be polarized to various subtypes including M1 and M2 in response to stimuli in their environment. M1 macrophages have anti-tumor functions such as immune stimulation. M2 macrophages have pro-tumor functions such as promoting angiogenesis, extracellular matrix remodeling and immune suppression. The majority of prostate cancer tumor-associated macrophages exhibit M2-like characteristics. Thus, targeting M2 tumor-associated macrophages is a promising strategy for cancer therapy. M2 macrophages engage in efferocytosis, or phagocytosis of apoptotic cells. This process is an anti-inflammatory, immunosuppressive event that promotes tissue remodeling and tumor growth. The TAM receptors (Tyro3, Axl, MerTK) are a family of receptor tyrosine kinases whose role in efferocytosis has been well described in the literature. The TAM receptors bind phosphatidylserine on apoptotic cells using their ligands Gas6 and Protein S as bridging proteins. Following binding to phosphatidylserine, TAM receptor signaling induces a cytoskeleton rearrangement to engulf the apoptotic cell. Since efferocytosis is a tumor promoting process, we hypothesize targeting the TAM receptors on M2 macrophages may be a beneficial anti-cancer strategy. We are characterizing TAM receptor expression on M1 and M2 macrophages using two complementary in vitro methods: macrophage polarization of monocytes derived from healthy human donors and of the acute monocytic leukemia cell line THP-1. Cell surface markers of M1 macrophages (CD86) and M2 macrophages (CD163 and CD206) were measured to assess polarization by flow cytometry. We are currently further confirming the degree of polarization by measuring cytokine secretion and mRNA expression of M1- and M2-associated genes. We used flow cytometry analysis to assess TAM receptor expression on the in vitro polarized M1 and M2 macrophages. In the human monocyte derived model, we detected higher levels of expression of MerTK and Tyro3 on M2 macrophages in several biological replicates. We also found that M2 THP-1 macrophages express higher levels of MerTK and Tyro3 than M1 THP-1 macrophages, consistent with macrophages from the monocyte derived model. In conclusion, these data support the TAM receptors as a relevant therapeutic target for inhibiting M2 pro-tumor functions. Further work by qRT-PCR, western blot and immunofluorescence staining will be used to confirm higher levels of the MerTK and Tyro3 on M2 macrophages and further investigate expression of Axl on M1 and M2 macrophages. Citation Format: Kayla V. Myers, Kenneth J. Pienta. Targeting the TAM receptors on prostate cancer tumor-associated macrophages [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4556.

Abstract 2360: Targeting IL-4R alpha on tumor-associated macrophages as a therapeutic strategy for prostate cancer

Abstract Prostate cancer tumor growth and disease progression are highly influenced by non-cancerous host cells within the tumor microenvironment including immune cells. High prevalence of tumor-associated macrophages (TAMs) in tumor biopsies is associated with poorer outcomes. TAMs comprise as much as 50 percent of prostate cancer tumors making them an abundant and critical target for treating prostate cancer. Macrophages adopt different functions in response to signaling molecules in their environment. The majority of prostate cancer TAMs resemble alternatively-activated M2 macrophages which, in healthy individuals, are primarily involved in wound healing. M2 TAMs behave similarly to wound-healing M2s by promoting cancer progression and immune suppression. Macrophages are converted to M2s when the signaling molecules interleukin-4 (IL-4) or IL-13 bind IL-4 Receptor Type I or II. Both of these receptor complexes contain the IL-4 receptor alpha subunit and signal through STAT6 to regulate downstream gene transcription and conversion to M2. Due to the central role of IL-4R alpha in tumor-promoting M2s, we hypothesize that disrupting IL-4R alpha signaling will undermine the tumor-promoting capabilities of M2-like TAMs. Using human macrophages cultured in vitro, we detect IL-4R alpha expression in non-malignant human macrophages by qPCR and immunoblot, critical data that is absent in the current literature. Additionally, using the FDA-approved antibody dupilumab, we show that antagonizing IL-4R alpha prevents STAT6 phosphorylation both before and after macrophages have been converted to M2s. Additionally, we show that dupilumab mitigates the M2 characteristics of these macrophages by nanostring analysis of M2-associated genes and ELISA of immunosuppressive cytokines. These data provide evidence that targeting IL-4R alpha on human macrophages impairs tumor-promoting M2 functions. To assess IL-4R alpha expression on TAMs in vivo, we used the HiMyc and TRAMP transgenic mouse models. These mice develop spontaneous prostate cancer tumors and are more accurate representations of the primary tumor microenvironment than injected tumor models (i.e. subcutaneous and orthotopic models). Using nanostring analysis and immunohistochemistry of prostates from 8-week-, 6-month-, and 12-month-old mice, we assessed immune cell infiltration levels, M2 characteristics of TAMs, and IL-4R alpha expression of TAMs. This data provides evidence for the M2-like characteristics of TAMs and IL-4R alpha as a viable TAM target. Subsequent experiments will include assessing IL-4R alpha expression on TAMs in human tumors using prostate cancer tissue microarrays. Moreover, we will continue to investigate the therapeutic potential of antagonizing IL-4R alpha through in vivo studies. In conclusion, these data implicate targeting IL-4R alpha as a promising therapeutic strategy for targeting TAMs in prostate cancer. Citation Format: Amber E. De Groot, Kayla Myers, Sarah R. Amend, Kenneth J. Pienta. Targeting IL-4R alpha on tumor-associated macrophages as a therapeutic strategy for prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2360.

RNAseq Profiling of Leukocyte Populations in Zebrafish Larvae Reveals a cxcl11 Chemokine Gene as a Marker of Macrophage Polarization During Mycobacterial Infection.

Macrophages are phagocytic cells from the innate immune system, which forms the first line of host defense against invading pathogens. These highly dynamic immune cells can adopt specific functional phenotypes, with the pro-inflammatory M1 and anti-inflammatory M2 polarization states as the two extremes. Recently, the process of macrophage polarization during inflammation has been visualized by real time imaging in larvae of the zebrafish. This model organism has also become widely used to study macrophage responses to microbial pathogens. To support the increasing use of zebrafish in macrophage biology, we set out to determine the complete transcriptome of zebrafish larval macrophages. We studied the specificity of the macrophage signature compared with other larval immune cells and the macrophage-specific expression changes upon infection. We made use of the well-established mpeg1, mpx, and lck fluorescent reporter lines to sort and sequence the transcriptome of larval macrophages, neutrophils, and lymphoid progenitor cells, respectively. Our results provide a complete dataset of genes expressed in these different immune cell types and highlight their similarities and differences. Major differences between the macrophage and neutrophil signatures were found within the families of proteinases. Furthermore, expression of genes involved in antigen presentation and processing was specifically detected in macrophages, while lymphoid progenitors showed expression of genes involved in macrophage activation. Comparison with datasets of in vitro polarized human macrophages revealed that zebrafish macrophages express a strongly homologous gene set, comprising both M1 and M2 markers. Furthermore, transcriptome analysis of low numbers of macrophages infected by the intracellular pathogen Mycobacterium marinum revealed that infected macrophages change their transcriptomic response by downregulation of M2-associated genes and overexpression of specific M1-associated genes. Among the infection-induced genes, a homolog of the human CXCL11 chemokine gene, cxcl11aa, stood out as the most strongly overexpressed M1 marker. Upregulation of cxcl11aa in Mycobacterium-infected macrophages was found to require the function of Myd88, a critical adaptor molecule in the Toll-like and interleukin 1 receptor pathways that are central to pathogen recognition and activation of the innate immune response. Altogether, our data provide a valuable data mining resource to support infection and inflammation research in the zebrafish model.

Activated Macrophages of Monocytic Origin Predominantly Express Proinflammatory Cytokine Genes, Whereas Kupffer Cells Predominantly Express Anti-Inflammatory Cytokine Genes.

In the central nervous system and in the liver, the macrophage populations are represented exclusively by descendants of the hematopoietic progenitor cells of the yolk sac. The reasons for such differential distribution of macrophages are not fully understood. We found that, as can be judged by corresponding changes in the expression of CD86 and CD163 markers, the transient macrophages of monocytic lineage are more sensitive to activating stimuli. The two macrophage populations have distinct patterns of gene expression, which is particularly noticeable for M1- and M2-associated genes. For instance, Kupffer cells more readily develop and longer maintain the elevated expression levels of Il4, Il10, and Il13 upon the activation; by contrast, the macrophages of monocytic lineage express Il1b, Il12a, and Tnfα upon the activation. The obtained results allow us to conclude that the in vitro activated Kupffer cells of the liver are committed to M2 phenotype, whereas the in vitro activated monocyte-derived macrophages show a typical M1 behavior. These observations are likely to reflect the situation in the in vivo microenvironments.

Identification of alterations in macrophage activation associated with disease activity in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is characterized by abnormalities in B cell and T cell function, but the role of disturbances in the activation status of macrophages (Mϕ) has not been well described in human patients. To address this, gene expression profiles from isolated lymphoid and myeloid populations were analyzed to identify differentially expressed (DE) genes between healthy controls and patients with either inactive or active SLE. While hundreds of DE genes were identified in B and T cells of active SLE patients, there were no DE genes found in B or T cells from patients with inactive SLE compared to healthy controls. In contrast, large numbers of DE genes were found in myeloid cells (MC) from both active and inactive SLE patients. Among the DE genes were several known to play roles in Mϕ activation and polarization, including the M1 genes STAT1 and SOCS3 and the M2 genes STAT3, STAT6, and CD163. M1-associated genes were far more frequent in data sets from active versus inactive SLE patients. To characterize the relationship between Mϕ activation and disease activity in greater detail, weighted gene co-expression network analysis (WGCNA) was used to identify modules of genes associated with clinical activity in SLE patients. Among these were disease activity-correlated modules containing activation signatures of predominantly M1-associated genes. No disease activity-correlated modules were enriched in M2-associated genes. Pathway and upstream regulator analysis of DE genes from both active and inactive SLE MC were cross-referenced with high-scoring hits from the drug discovery Library of Integrated Network-based Cellular Signatures (LINCS) to identify new strategies to treat both stages of SLE. A machine learning approach employing MC gene modules and a generalized linear model was able to predict the disease activity status in unrelated gene expression data sets. In summary, altered MC gene expression is characteristic of both active and inactive SLE. However, disease activity is associated with an alteration in the activation of MC, with a bias toward the M1 proinflammatory phenotype. These data suggest that while hyperactivity of B cells and T cells is associated with active SLE, MC potentially direct flare-ups and remission by altering their activation status toward the M1 state.

Astragaloside IV inhibits lung cancer progression and metastasis by modulating macrophage polarization through AMPK signaling

BackgroundAccumulating evidence suggests that M2-polarized tumor-associated macrophages (TAMs) play an important role in cancer progression and metastasis, making M2 polarization of TAMs an ever more appealing target for therapeutic intervention. Astragaloside IV (AS-IV), a saponin component isolated from Astragali radix, has been reported to inhibit the invasion and metastasis of lung cancer, but its effects on TAMs during lung cancer progression have not been investigated.MethodsHuman THP-1 monocytes were induced to differentiate into M2 macrophages through treatments with IL-4, IL-13, and phorbol myristate acetate (PMA). We used the lung cancer cell lines A549 and H1299 cultured in conditioned medium from M2 macrophages (M2-CM) to investigate the effects of AS-IV on tumor growth, invasion, migration, and angiogenesis of lung cancer cells. Macrophage subset distribution, M1 and M2 macrophage-associated markers, and mRNA expression were analyzed by flow cytometry and quantitative PCR. The activation of adenosine monophosphate-activated protein kinase (AMPK) signaling pathways that mediate M2-CM–promoted tumor migration was detected using western blotting.ResultsHere we found that AS-IV significantly inhibited IL-13 and IL-4–induced M2 polarization of macrophages, as illustrated by reduced expression of CD206 and M2-associated genes, and that AS-IV suppressed the M2-CM–induced invasion, migration, and angiogenesis of A549 and H1299 cells. In vivo experiments demonstrated that AS-IV greatly inhibited tumor growth and reduced the number of metastases of Lewis lung cancer. The percentage of M2 macrophages was decreased in tumor tissue after AS-IV treatment. Furthermore, AS-IV inhibited AMPKα activation in M2 macrophages, and silencing of AMPKα partially abrogated the inhibitory effect of AS-IV.ConclusionsAS-IV reduced the growth, invasion, migration, and angiogenesis of lung cancer by blocking the M2 polarization of macrophages partially through the AMPK signaling pathway, which appears to play an important role in AS-IV’s ability to inhibit the metastasis of lung cancer.

Abstract 3471: Expression of the DEK oncogene in breast cancer cells promotes M2 polarization of tumor associated macrophages

Abstract Breast cancer is the most common cancer and the second leading cause of cancer deaths among women. DEK is a known oncoprotein found to be highly expressed in more than 60% breast cancers and is found to be an independent marker of poor prognosis. However, the molecular mechanisms by which DEK promotes tumor progression are poorly understood. To elucidate the oncogenic functions of DEK, we performed RNA-seq analysis on isogenic Dek-knockout and complemented murine breast cancer cells, which indicated dysregulation of immune signaling. Among the target genes identified and confirmed was an upregulation of thymic stromal lymphopoietin (TSLP) in Dek expressing breast cancer cells. TSLP was previously shown by Han et al to amplify the alternative (M2) activation of macrophages. M2-like macrophages are tumor promoting, they recycle iron for cell growth and encourage tissue remodeling and repair activities like angiogenesis. Classically (M1) activated macrophages are tumor suppressing and are typically associated with response to infection. To test the immune modulating functions of Dek-expressing breast cancer cells, we treated bone marrow derived macrophages (BMDM) with tumor cell conditioned media. We found that in vitro, Dek expressing cancer cells induced an M2-like polarization of macrophages, as determined by the expression of M2-associated genes, enhanced migration, and iron recycling phenotypes, which was accompanied by inhibited ERK1/2 sighnaling. We found this phenotypic trend to be true in vivo, as well. In sectioned mammary tumors from MMTV-RontgDek+/+ and MMTV-RontgDek-/- mice, we see lower levels of iron staining in Dek+/+ tumors than in Dek-/- tumors using Prussian blue staining that co-localized with F4/80 macrophage marker staining. This suggested that Dek expression in the tumors induced an iron recycling, M2-like phenotype in tumor associated macrophages. These findings suggest that tumor Dek expression may promote breast cancer progression by inducing M2-like macrophage polarization in a murine breast cancer model. Citation Format: Miranda S. Shephard, Nicholas A. Pease, Jon Cheek, Lisa M. Privette Vinnedge. Expression of the DEK oncogene in breast cancer cells promotes M2 polarization of tumor associated macrophages [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3471.

Identification of perturbations in macrophage polarization in active systemic lupus erythematosus.

Abstract SLE is characterized by abnormalities in B cell and T cell function, but the role of disturbances in the polarization of macrophages (Mφ) into functional subpopulations is unclear. Gene expression profiles were analyzed to identify differentially expressed (DE) genes between healthy controls and patients with either inactive or active SLE. While hundreds of DE genes were identified in B and T cells of active SLE patients, there were no DE genes found in B or T cells from patients with inactive SLE compared to healthy controls. In contrast, large numbers of DE genes were found in myeloid cells (MC) from both active and inactive SLE patients, and many of these were Mφ polarization genes (M1: STAT1, SOCS3; M2: STAT3, STAT6, CD163). Weighted gene correlation network analysis (WGCNA) was used to identify modules of genes associated with clinical activity in SLE patients; among these were disease activity-correlated modules containing polarization signatures of predominantly M1-associated genes. No disease activity-correlated modules were enriched in M2-associated genes. Pathway and upstream regulator analysis of DE genes from both active and inactive SLE MC were cross-referenced with high-scoring hits from the drug discovery Library of Integrated Network-based Cellular Signatures (LINCS) to identify new strategies to treat both stages of SLE. In summary, altered MC gene expression is characteristic of both active and inactive SLE, and disease activity is associated with an alteration in the MC signature towards the M1 phenotype. These data suggest that while B and T cell hyperactivity are associated with active SLE pathology, myeloid cells potentially direct flare-ups and remission by switching between M1- and M2-like polarization states.

Macrophages in keloid are potent at promoting the differentiation and function of regulatory T cells

The mechanistic details of keloid formation are still not understood. Given that the immune system is engaged in skin lesion repair, we examined the CD14+ macrophages and CD3+ T cells in keloid tissues and in the normal skin. Compared to the normal skin, keloid tissues presented significantly elevated infiltration by CD14+ macrophages. Moreover, the transcription and protein expression of iNOS, IL-12, IL-10, and TGF-β were significantly higher in keloid macrophages than in normal skin macrophages, in which the expression of M2-associated genes were further elevated compared to M1-associated genes in keloid. We also observed that keloid tissues presented higher infiltration by CD3+ T cells, of which the majority was CD4+ T cells. Notably, the frequency of Foxp3+ regulatory T cells (Tregs) in keloid tissues was significantly higher compared to that in the peripheral blood. Furthermore, macrophages from keloid tissues possessed potent capacity to induce Foxp3 expression in circulating CD3+ T cells. Together, this study suggested that macrophages in keloid tissues presented high activation status and were polarized toward the M2 subtype; moreover, these macrophages could promote Treg differentiation by upregulating Foxp3 expression.

Regulation of Microglia and Macrophage Polarization via Apoptosis Signal-Regulating Kinase 1 Silencing after Ischemic/Hypoxic Injury.

Inflammation is implicated in ischemic stroke and is involved in abnormal homeostasis. Activation of the immune system leads to breakdown of the blood–brain barrier and, thereby, infiltration of immune cells into the brain. Upon cerebral ischemia, infiltrated macrophages and microglia (resident CNS immune cell) are activated, change their phenotype to M1 or M2 based on the microenvironment, migrate toward damaged tissue, and are involved in repair or damage. Those of M1 phenotype release pro-inflammatory mediators, which are associated with tissue damage, while those of M2 phenotype release anti-inflammatory mediators, which are related to tissue recovery. Moreover, late inflammation continually stimulates immune cell infiltration and leads to brain infarction. Therefore, regulation of M1/M2 phenotypes under persistent inflammatory conditions after cerebral ischemia is important for brain repair. Herein, we focus on apoptosis signal-regulating kinase 1 (ASK1), which is involved in apoptotic cell death, brain infarction, and production of inflammatory mediators after cerebral ischemia. We hypothesized that ASK1 is involved in the polarization of M1/M2 phenotype and the function of microglia and macrophage during the late stage of ischemia/hypoxia. We investigated the effects of ASK1 in mice subjected to middle cerebral artery occlusion and on BV2 microglia and RAW264.7 macrophage cell lines subjected to oxygen-glucose deprivation. Our results showed that ASK1 silencing effectively reduced Iba-1 or CD11b-positive cells in ischemic areas, suppressed pro-inflammatory cytokines, and increased anti-inflammatory mediator levels at 7 days after cerebral ischemia. In cultured microglia and macrophages, ASK1 inhibition, induced by NQDI-1 drug, decreased the expression and release of M1-associated factors and increased those of M2-associated factors after hypoxia/reperfusion (H/R). At the gene level, ASK1 inhibition suppressed M1-associated genes and augmented M2-associated genes. In gap closure assay, ASK1 inhibition reduced the migration rate of microglia and macrophages after H/R. Taken together, our results provide new information that suggests ASK1 controls the polarization of M1/M2 and the function of microglia and macrophage under sustained-inflammatory conditions. Regulation of persistent inflammation via M1/M2 polarization by ASK1 is a novel strategy for repair after ischemic stroke.

Abstract 450: Monocytic Glutaredoxin 1 Protects Mice Against Obesity, Hyperglycemia and Atherosclerosis

Overexpression of glutaredoxin 1 (Grx1) protects monocytes from metabolic stress-induced priming, i.e. dysregulation and hypersensitization to chemokines (Ullevig et al. ATVB 2012). To address the role of monocytic Grx1 in mice and in the development of atherogenesis and obesity, we transplanted bone marrow (BM) from either wild-type (WT) or Grx1 -/- donor mice into atherosclerosis-prone LDLR -/- mice and fed these mice a high-fat diet (HFD) for up to 20 weeks. Grx1 Leuko -/- mice showed accelerated weight gain after 9 weeks followed by early onset of hyperglycemia. After 6 weeks on HFD, atherosclerotic lesions were slightly larger in Grx1 Leuko -/- mice than in WT mice, but the differences did not reach statistical significance. However, after 20 weeks, Grx1 Leuko -/- mice showed 36% larger lesions than WT-BM recipients, and monocyte chemotaxis in vivo was increased 1.6-fold. Furthermore, compared to WT-BM recipients, adipose tissues and livers of Grx1 Leuko -/- mice also showed increased macrophage content and elevated tissue inflammation as determined by IHC and qRT-PCR-based gene array. Adipose tissue in particular, showed significant increases in the expression of proinflammatory genes in addition to an increased abundance of proinflammatory “crown-like” structures. In contrast, genes associated with inflammation resolving macrophages were significantly suppressed. Macrophages isolated from Grx1 -/- mice and stimulated with INFγ+TNFα also showed increased expression of pro-inflammatory M1-associated genes, whereas M2-associated genes were suppressed in Grx-1 -/- macrophages activated with IL-4. Furthermore, macrophages from Grx1 -/- mice exposed to metabolic stress also display increased protein S -glutathionylation, enhanced hypersensitization to chemokine, and impaired autophagy compared to macrophages from wild-type mice. Taken together, our data show that loss of monocytic Grx1 worsens monocyte priming in response to HFD-induced metabolic stress and accelerates the infiltration of dysfunctional monocyte-derived macrophages into tissues, such as aorta, liver and adipose tissues. We conclude that monocytic Grx1 is critical for maintaining metabolic homeostasis in mice and protects mice against obesity and atherogenesis.

Identification of perturbations in macrophage polarization in active systemic lupus erythematosus.

Abstract SLE is characterized by abnormalities in B cell and T cell function, but the role of disturbances in the polarization of macrophages (Mφ) into functional subpopulations has not been delineated. Gene expression profiles were analyzed to identify differentially expressed (DE) genes between healthy controls and patients with either inactive (SLEDAI < 6) or active (SLEDAI ≥ 6) SLE. While hundreds of DE genes were identified in B and T cells of active SLE patients (760 and 164 genes respectively), there were no DE genes found in B or T cells from patients with inactive SLE compared to healthy controls. In contrast, large numbers of DE genes were found in myeloid cells from both active (2,135) and inactive (1,260) SLE patients. Many of these DE genes were known to play roles in Mφ polarization, including the M1 genes STAT1 and SOCS3 and the M2 genes STAT3, STAT6, and CD163. M1-associated genes were far more frequent in data sets from active versus inactive SLE patients. To further characterize the relationship between M1 phenotype and disease activity, Weighted Gene Cluster Network Analysis (WGCNA) was used to identify modules of genes associated with clinical activity in SLE patients. Among these were disease activity-correlated modules containing polarization signatures of predominantly M1-associated genes. No disease activity-correlated modules were enriched in M2-associated genes. In summary, altered Mφ gene expression is characteristic of both active and inactive SLE. However, disease activity is associated with an alteration in the functional differentiation of Mφ to the M1 proinflammatory phenotype. This switch in Mφ phenotype is related to the altered expression of genes by T cells and B cells characteristic of active SLE.

Transcriptome analysis of microglia in a mouse model of Rett syndrome: differential expression of genes associated with microglia/macrophage activation and cellular stress

BackgroundRett syndrome (RTT) is a severe, neurodevelopmental disorder primarily affecting girls, characterized by progressive loss of cognitive, social, and motor skills after a relatively brief period of typical development. It is usually due to de novo loss of function mutations in the X-linked gene, MeCP2, which codes for the gene expression and chromatin regulator, methyl-CpG binding protein 2. Although the behavioral phenotype appears to be primarily due to neuronal Mecp2 deficiency in mice, other cell types, including astrocytes and oligodendrocytes, also appear to contribute to some aspects of the RTT phenotype. In addition, microglia may also play a role. However, the effect of Mecp2 deficiency in microglia on RTT pathogenesis is controversial.MethodsIn the current study, we applied whole transcriptome analysis using RNA-seq to gain insight into molecular pathways in microglia that might be dysregulated during the transition, in female mice heterozygous for an Mecp2-null allele (Mecp2+/−; Het), from the pre-phenotypic (5 weeks) to the phenotypic phases (24 weeks).ResultsWe found a significant overlap in differentially expressed genes (DEGs) with genes involved in regulating the extracellular matrix, and those that are activated or inhibited when macrophages and microglia are stimulated towards the M1 and M2 activation states. However, the M1- and M2-associated genes were different in the 5- and 24-week samples. In addition, a substantial decrease in the expression of nine members of the heat shock protein (HSP) family was found in the 5-week samples, but not at 24 weeks.ConclusionsThese findings suggest that microglia from pre-phenotypic and phenotypic female mice are activated in a manner different from controls and that pre-phenotypic female mice may have alterations in their capacity to response to heat stress and other stressors that function through the HSP pathway.

Differential response to intrahippocampal interleukin-4/interleukin-13 in aged and exercise mice

Normal aging is associated with low-grade neuroinflammation that results from age-related priming of microglial cells. Further, aging alters the response to several anti-inflammatory factors, including interleukin (IL)-4 and IL-13. One intervention that has been shown to modulate microglia activation in the aged brain, both basally and following an immune challenge, is exercise. However, whether engaging in exercise can improve responsiveness to anti-inflammatory cytokines is presently unknown. The current study evaluated whether prior exercise training increases sensitivity to anti-inflammatory cytokines that promote the M2 (alternative) microglia phenotype in adult (5-month-old) and aged (23-month-old) C57BL/6J mice. After 8weeks of exercise or control housing, mice received bilateral hippocampal injections of an IL-4/IL-13 cocktail or vehicle. Twenty-four hours later hippocampal samples were collected and analyzed for expression of genes associated with the M1 (inflammatory) and M2 microglia phenotypes. Results show that IL-4/IL-13 administration increased expression of the M2-associated genes found in inflammatory zone 1 (Fizz1), chitinase-like 3 (Ym1), Arginase-1 (Arg1), SOCS1, IL-1ra, and CD206. In response to IL-4/IL-13 administration, aged mice showed increased hippocampal expression of the M2-related genes Arg1, SOCS1, Ym1, and CD206 relative to adult mice. Aged mice also showed increased expression of IL-1β relative to adults, which was unaffected by wheel running or IL-4/IL-13. Wheel running was found to have modest effects on expression of Ym1 and Fizz1 in aged and adult mice. Collectively, our findings indicate that aged mice show a differential response to anti-inflammatory cytokines relative to adult mice and that exercise has limited effects on modulating this response.

Characterization of peritoneal leukemia-associated macrophages in Notch1-induced mouse T cell acute lymphoblastic leukemia.

Macrophages, which have remarkable plasticity, are indispensable cellular components and play essential roles in both innate and adaptive immune responses. Peritoneal macrophages show unique gene expression profile and peritoneal cavity is also involved in leukemia. However, the characteristics of peritoneal leukemia-associated macrophages (Per LAMs) have not been established. Here we studied the phenotype of Per LAMs, their subpopulations in Notch1-induced acute lymphoblastic leukemia mice and compared with LAMs from BM or spleen in the same model. Peritoneal macrophages and Per LAMs simultaneously expressed high level iNOS and Arg1, which was not commonly observed in macrophages from different origins. Furthermore, LAMs from peritoneal, BM and spleen expressed lower level CSF-1, TGF-β1 and VEGF-A than tumor-associated macrophages (TAMs). Moreover, diverse responses in the expression of some phenotype-associated genes to leukemia microenvironments were detected among those LAMs. In addition, Per LAMs can be sub-divided into CD206+ and CD206- sub-populations, which expressed both M1- and M2-associated genes. These results revealed the unique phenotype of Per macrophages and Per LAMs and contributed to better understanding of macrophage plasticity and their pathological roles in leukemia.

96 - Monocytic Glutaredoxin 1 Protects Mice against Obesity, Hyperglycemia and Atherosclerosis

We previously reported that glutaredoxin 1 (Grx1) protects monocytes from metabolic-stress induced priming, dysregulation and hypersensitization to chemokines. To address the role of monocytic Grx1 in mice and in the development of atherogenesis and obesity, we transplanted bone marrow (BM) from either wild-type (WT) or Grx1-/- (Grx1Leuko -/-) donor mice into atherosclerosis-prone LDLR-/- mice and fed these mice a high-fat diet (HFD) for up to 20 weeks. Grx1Leuko -/- mice showed accelerated weight gain after 9 weeks followed by early onset of hyperglycemia. After 6 weeks on HFD, atherosclerotic lesions were slightly larger in Grx1Leuko -/- mice than in WT, but the differences did not reach statistical significance. However, after 20 weeks, Grx1Leuko -/- mice showed 36% larger lesions than WT-BM recipients, and monocyte chemotaxis in vivo was increased 1.6-fold. Furthermore, compared to WT-BM recipients, adipose tissues and livers of Grx1Leuko -/- mice also showed increased macrophage content and elevated tissue inflammation as determined by IHC and qRT-PCR. Adipose tissue in particular, showed significant increases in the expression of proinflammatory genes in addition to an increased abundance proinflammatory “crown-like” structures. Gene expression analysis by qRT-PCR of peritoneal macrophages revealed that macrophages isolated from Grx1-/- mice also have increased expression of pro-inflammatory M1-associated genes and decreased M2-associated genes after activation with INFγ+TNFα and IL-4 respectively. Additionally, macrophages from Grx1-/- mice exposed to metabolic stress also display increased protein-S-glutathionylation, enhanced hypersensitization to chemokine, and impaired autophagy compared to wild-type cells. Taken together, our data show that loss of monocytic Grx1 worsens monocyte priming and accelerates the infiltration of dysfunctional monocyte-derived macrophages into tissues, such as aorta, liver and adipose tissues. We conclude that monocytic Grx1 is critical for maintaining metabolic homeostasis in mice and protects against obesity and atherosclerosis.